I have shown conclusively that at least two of the major outer membrane proteins from Escherichia coli K-12 contain allysine (alpha-aminoadipic acid gamma-semialdehyde) and it was found to be derived from lysine. This compound functions in connective tissue as the aldehyde donor in the formation of lysyl-derived aldehydic cross-links. It is the purpose of this proposal to determine the kinds of aldehydic cross-links present in the outer membrane of E. coli, the extent of allysine formation in the outer membrane, and the physiological conditions which influence the extent and distribution of lysine-derived cross-links in the outer membrane. Experiments will be performed to evaluate the function of these cross-links on the organization, stability, permeability, and assembly of the gram-negative bacterial envelope. Experimentally, aldehydic cross-links will be isolated from cell envelopes as stabilized derivatives obtained by chemical reduction of the cross-link or by employing a modified Strecker reaction to stabilize the cross-link. This material will be purified and tentatively identified on an amino acid analyzer, and final identification will be by mass spectrometry. Experiments will also be performed to purify and characterize the enzyme responsible for the conversion of lysine to allysine in E. coli.